Abstract Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) with ~108 million annual cases worldwide and ~1.44 million U.S. cases in 2014. Most female and male infections are asymptomatic, facilitating unchecked transmission that can result in pelvic inflammatory disease, infertility, chronic pelvic pain, and life-threatening ectopic pregnancy. Ct is also a risk factor for invasive squamous-cell carcinoma of the cervix and a complicating factor in HIV-1 infection. Patients are often treated without a definitive diagnosis, which can lead to inappropriate antibiotic use and possibly drug resistance. The main obstacle to stemming Ct infections is the lack of a point-of-care (POC) diagnostic to increase early detection to reduce infection rates and sequelae. Current Ct diagnostics rely on commercial nucleic acid amplification tests (NAATs) that vary in sensitivity and specificity with a general lack of concordant results for the same sample type. NAATs are expensive, require equipment and highly trained operators, take a day to days for results, and can result in loss to follow up and delay in treatment. Thus, current NAATs are not suitable POC diagnostics. Our team of Dr. Deborah Dean, an expert on Ct STDs and POC development, and Diassess, a startup company with proprietary technology for POC diagnostics, showed in Phase I that we: 1) can extract Ct nucleic acids from endocervical swabs in 5 min with no instruments in a prototype Sample Preparation Module; 2) have an instrument-free multiplexed Detection Module for 30 min colorimetric detection of Ct nucleic acids; 3) have validated assays to detect all Ct reference strains, differentiate lymphogranuloma venereum (LGV) from non-LGV strains and detect human DNA; and 4) have demonstrated assay results consistent with standard NAAT results on 200 remnant endocervical patient samples. In Phase II, we will advance on Phase I results. Aim 1: Using the expanding aggregate of reference and clinical Ct genome sequences, refine our primers, replacing failed primers as needed and ensure that our refined Ct primer amplification assays detect Ct reference and diverse Ct clinical strains without cross-reactivity with sexually transmitted pathogens and common vaginal/cervical species; Aim 2: Optimize sample preparation chemistry, amplification assay design and colorimetric chemistry for vaginal, urethral and endocervical swabs, and interfering substances; Aim 3: Evaluate sensitivity and specificity of our fully-integrated system (the combined Sample Preparation Module with Detection Module) compared to commercial NAATs. By the end of Phase II, we will be poised to manufacture and use our rapid (<35 min), inexpensive, user-friendly, instrument-free, sensitive and specific Ct POC test for clinical trials in the U.S. to obtain FDA regulatory clearance via the 510(k) pathway. Our overall goal is to deploy our Ct POC diagnostic for use in doctor's offices, small to large city and rural clinics, teen and STD clinics, emergency rooms, other testing sites and resource-constrained settings around the world.